Caulobacter crescentus is an asymmetrically dividing, Gram negative bacterium that has proved to be an excellent model for the study of cell differentiation. The most striking feature of the stalked cell cycle of this organism is the stage specific sequence of events that leads to the generation of a new motile swarmer cell at division. Our long term goal is to understand the developmental program that controls the precise sequence of these events and coordinates their timing and localization of cell surface structures. Our studies have focused on flagellum formation which requires over 50 genes that are organized in a regulatory hierarchy. Periodic expression of the flagellar genes in the cell cycle is mediated by a cascade of trans-acting regulatory factors. The goal of work proposed in this application is to characterize the trans-acting factors and the DNA sequence elements with which they interact to determine the observed temporal regulation of transcriptional specificity. Our specific aims are to i) Determine the location and mechanism of FlbD binding to DNA sequence elements required for regulation of sigma 54 promoters; ii) Determine if FlbD, which is a functional homologue of response regulatory protein NtrC, is modified by phosphorylation and the role of modification for its function as a transcriptional factor; iii) Identify genes in the flaO and flbF operons that are required along with flbD for activation of sigma 54 promoters at the bottom of the hierarchy; iv) Purify RNA polymerases from Caulobacter to characterize specialized sigma factors in order to asses their role in determining promoter specificity; v) Investigate the role of DNA synthesis as a cell cycle signal that triggers the flagellar gene cascade; and vi) Examine the hook protein for sequences that target it to the cell pole for assembly using translation fusions to reporter genes.